Drugs of choice used in the treatment of pulmonary hypertension (PH) clearly show that endothelin-1 (ET-1) is a major participant in this fatal disease. Angiotensin (AngII) through its AT1 receptor stimulates the production of ET-1 in human lung vascular endothelial cells (HLVEC). ET-1 then stimulates the production of vasodilating, antiproliferative nitric oxide (NO) and prostacyclin (PGI2) through its ETB receptor in the HLVEC. In human lung vascular smooth muscle cells (HLVSMC) ET-1 stimulates cell proliferation and vasoconstriction. In PH this homeostatic regulation breaks down. The AT1 receptor, which is involved at the initial stage of PH, propagates many of the signals participating in the etiology of PH. Our studies on receptor signaling motifs combined with mutant receptor transfection and signal cascade determinations have enabled us to control the signaling of the AT1 receptor. More recently we can now control signaling by the (wild type) endogenously expressed receptors with the use of cell penetrating peptides constructed to mimic the receptor motifs sequence(s). We intend to achieve signal control in the ETA and ETB receptors and then regulate these signal transductions in human lung vascular endothelial and smooth muscle cells. It is anticipated that this approach will lead to new treatments of PH. Our working hypothesis is that signal cascades involving MAPKs, the Akt/PI3K/PTEN system, small G-protein RhoA and heterotrimeric G- proteins work in various combinations to produce ET-1 through the AT1 receptor and to produce NO and PGI2 through the ETB receptor in human lung vascular endothelial cells and to produce contraction and proliferation through the ETA/ETB receptors in human lung vascular smooth muscle cells; that these signal interactions can be controlled through the use of altered receptors and cell penetrating peptides; and that by altering these signals, we will be able to decrease ET-1 and increase NO and prostacyclin production by endothelial cells and decrease growth and contraction in smooth muscle cells. Specific aims, 1) a) We will regulate AT1, ETA and ETB receptor signaling through motif identification and mutation construction within the receptor to regulate G1q, G1i, RhoA, Akt and MAPKs. b) We will then regulate signaling in endogenously expressed AT1 and ETB receptors in human lung endothelial cells and ETA receptors in smooth muscle cells with use of membrane permeable peptides. 2) With use of the altered receptors and the peptides developed in SA 1, we will regulate the expression and release of ET-1 by AngII and the release of NO and prostacyclin in the HLVEC by ET-1. These results will then be used to control function of these receptors in HLVEC isolated from lung transplants of individuals with PH. 3) With use of the altered receptors and the peptides developed in Specific Aim 1, we will regulate contraction and proliferation in human lung vascular smooth muscle cells, including cells obtained from individuals with PH. 4) The studies on smooth muscle contraction in culture will be followed up with physiological experiments using rats. PUBLIC HEALTH RELEVANCE: Pulmonary hypertension is a debilitating and mostly fatal disease. Our experiments, including the production of peptides which interfere with negative signals of angiotensin and endothelin, are designed to lead to new approaches which should prove beneficial in the treatment of pulmonary hypertension.